A number of structural studies have confirmed the binding of sigma54 to RNAP and DNA. Sigma54 binds DNA -24 and -12 base pairs from the transcription start site to initiate DNA melting, an essential step in transcription. A substantial body of work, including DNA footprinting, alanine scanning mutation studies, low resolution cryoelectron microscopy of sigma54-RNA-polyermase bound to DNA in the closed configuration, and high resolution NMR structure has shown that sigma54 binds specifically and tightly to the major groove of DNA. For example the Wemmer lab used NMR to show that the 66 amino acid long helix-turn-helix (HTH) motif from in Aquifex aeolicus binds DNA. Within this C-terminal HTH motif, a single alpha-helix at residues 377 to 386 (ARRTVAKYRE), termed the RpoN box, is responsible for binding to the major groove of DNA (PBD: 2O8K). Replacement of Arg378, Arg379, Tyr384 and Arg385 with Ala decreased DNA binding substantially. The protein NMR structure shows that this helix interacts selectively with the -24 region (5'-TGGCACG-3') of the promoter. In particular, Arg378 and Arg379 are localized to the -24 element of the interaction and make multiple hydrogen bonding and ionic interactions with Gua-25 and Gua-26 on the non-coding strand of DNA based on significant line-broadening of a 15N-HSQC spectrum. This leads to a high affinity interaction between the 66-mer HTH motif and the promoter region (Kd = 114 nM) Although the peptides are modeled to inhibit transcription, it is necessary to assess cell viability to ensure that downstream antivirulence effects are observable. By using the standard broth microdilution method,24 the antimicrobial activity of all stapled peptide analogs was assessed. The minimum inhibitory concentration (MIC) value of all peptides is 32 microg/mL or higher. As this value is high compared to many conventional antibiotics, the peptide can be assessed at low concentrations to study its effects on virulence properties of Gram negative bacteria. To determine whether the compounds are cytotoxic to eukaryotic cells, WS1 fibroblasts were exposed to serial dilutions of each compound. The data demonstrate that the compounds are not toxic to normal cells over a concentration range peaking at 10 microM. In order to assess the helical structure of the synthesized peptides, circular dichroism spectroscopy was carried out by dissolving the compounds in water. While the wild type sigma54 peptide is unstructured, stapled peptides 1-4 all possess the hallmark spectrum of an alpha-helical secondary structure. Flow cytometry was used to gain a high-throughput perspective on the effectiveness of each stapled peptide in a large population of bacteria. By making use of its individual event detection, the percentage of cellular uptake of the peptides was determined. In both E. coli and P. aeruginosa species of Gram negative bacteria, all four peptide analogs are capable of penetration. Compared to the vehicle control, the wild type peptide, the sequence in which no staple is present, shows minor penetration in PA01 P. aeruginosa and insignificant levels of penetration in BW25113 E. coli. With each peptide, there is a penetration of at least 50% of cell in each peptide sample. In particular, sigma54-2 appears to have the highest penetrance whereas sigma54-4 has the least. To determine the mode of transport within the cell, sodium azide was used to inactivate ATPases used in hydrogen ion transport. All interior transport will be conducted through a non-active method such as passive or facilitated diffusion. With both types of treatments, it's possible to determine whether the mode of uptake of stapled peptides is through active or passive transport. Furthermore, with passive transport, the entry of the peptide should be faster than active due to the lack of metabolic means necessary to promote cell entry. The majority of peptide analogs increase in penetration upon sodium azide treatment. This may be an indicator that the peptide is able to be pumped out by bacteria to a degree. Overall, this assay demonstrates that stapled peptides are capable of penetrating cells better than their unstapled counterpart in a charge-independent manner. Further studies in confocal microscopy were conducted to gain a low-throughput visual method of assessing cell penetrance. The peptides correlate with the flow cytometry data in that some cells display strong fluorescence intensity and others display less. By using an image-based detection method for permeability, we observe that the double-stranded DNA of E. coli appears to aggregate in the center away from the membrane. In many of the cells observed, a number of the cells were undergoing cell division but still had significant uptake of these peptides. With the cell membranes, it is seen that the peptide does not integrate itself into the membrane due to a lack of green or masking of the red membrane stain. These peptides must cross both the cell wall and cell membrane to remain in the cytoplasm of bacteria. The binding of sigma54 RpoN with the -24 site of glnA was examined by performing a DNase protection assay which showed that stapled sigma 54 peptides protected DNA from degradation. To determine whether the peptides blocked the transcription of nitrogen metabolism genes, E. coli cells grown under nitrogen deficient conditions were treated with stapled peptides. RNA was isolated, and the sigma 54 dependent genes glnA, yeaG, and nac were analyzed. We also examined pspA which, despite being a sigma54 dependent gene, is insensitive to nitrogen depletion. The data showed that stapled sigma 54-2 and -3 were the best at blocking the transcription of sigma 54-dependent nitrogen depletion response genes. No effects were seen with pspA. Taken together, these results demonstrate that stapled peptides can be designed to target bacterial systems as well as protein-DNA interactions with a great level of specificity. The work on this project concluded in April 2019